1. Field of the Invention
The present invention relates to the creation of new classes of fluorescent/luminescent probes based on metal cluster fluorescence, methods of preparing such probes, and methods of use thereof.
2. Background Art
Single molecule fluorescence microscopy studies present an extreme limit in which weak signals must be observed on essentially zero background. Such optical methods relying on high-intensity laser excitation of highly emissive and robust fluorophores require extremely efficient background rejection. Relying on the introduction of artificial labels to identify the particular protein or structure of interest, fluorescence based methods suffer from two additional problems—photobleaching (loss of signal due to probe destruction) and autofluorescence (naturally occurring background fluorescence from native species within biological media). Even with these problems, fluorescence microscopy remains the primary optical method with potential for single molecule and chemical sensitivity while imaging biological media.
Most in vitro fluorescent labeling is performed through standard chemical coupling of either N-succinimidyl ester-conjugated dyes to free, solvent-exposed amines (often on lysine residues) or maleimide-conjugated dyes to thiols on either naturally occurring or genetically introduced solvent-exposed cysteines. These two coupling chemistries continue to be extremely useful in attaching small, highly fluorescent dyes to proteins of interest. Such fluorescent biomaterials are then adequate for in vitro single molecule studies, or they can be re-introduced into cells in high concentration either through microinjection or other membrane transport methods to perform bulk fluorescence studies of the protein of interest within whole cells. Not only can protein function be altered both by the size and point of attachment of the fluorescent label, but also often because of the coupling chemistry used, the site of the fluorescent labeling is not accurately known. Thus, the smallest possible genetically programmed label would be highly advantageous.
Additionally, in biological systems, the autofluorescent background from flavins, porphyrins, and all other weakly fluorescent naturally occurring species can produce a large background that interferes with laser-induced fluorescence signals. Because of these problems, studying dynamics of few copies of proteins within living systems requires the development of new fluorescent probes that absorb and emit so strongly and without significant photobleaching such that they can be easily observed with extremely weak incoherent illumination for long times. Such illumination would enable preferential excitation of the fluorophores of interest relative to that of weak background signals. Additionally, the weaker illumination would preserve biological viability by minimizing phototoxicity effects. Unfortunately, single molecule sensitivities are as of yet difficult to attain in such high background in vivo studies and are often difficult to observe even in lower background in vitro studies.
Because of signal to noise constraints, single molecule studies are limited to fluorescence-based assays with all its associated difficulties. While single molecule methods have been effective in “peeling back” the ensemble average to examine environmental and mechanistic heterogeneity, current techniques require expensive laser-based equipment, specialized synthetic methods, and are still fundamentally limited by the poor optical properties or bioincompatibility of available fluorescent labels.
Several key experiments have uniquely demonstrated the ability of single molecule microscopies to unravel the crucial steps leading to biological activity (Lu et al., Science 1998, 282:1877-1882; Dickson et al., Nature 1997, 388:355-358; Funatsu et al., Nature 1995, 374:555-59; Ha et al., Proc. Nat. Acad. Sci., USA 1996, 93, 6264-6268; Vale et al., Nature 1996, 380:451-453; Deniz et al., Proc. Natl. Acad. Sci., USA 2000, 97:5179-5184; Ha et al., Proc. Natl. Acad. Sci. USA 1999, 96:893-898; Harada et al., Biophys. J. 1999, 76:709-715; Kinosita, Biophys. J. 2000, 78:149Wkshp). Most dramatic in studies of individual motor protein motion, mechanistic insights into protein function and substeps within biomechanical cycles can be directly visualized, without the need of difficult external synchronization (Funatsu et al., Nature 1995, 374:555-59; Vale et al., Nature 1996, 380:451-453; Kinosita, Biophys. J. 2000, 78:149Wkshp; Yanagida et al., Curr. Opin. Cell Biol. 2000, 12:20-25). Even biomolecule folding has been probed to unravel pathways leading to both misfolded and folded states (Deniz et al., Proc. Natl. Acad. Sci., USA 2000, 97:5179-5184; Ha et al., Proc. Natl. Acad. Sci. USA 1999, 96:893-898). Now that orientational (Bartko, & Dickson, J. Phys. Chem. B 1999, 103:3053-3056; Bartko & Dickson, J. Phys. Chem. B 1999, 103:11237-11241; Bartko et al., Chem. Phys. Lett. 2002, 358:459-465; Hollars & Dunn, J. Chem. Phys. 2000, 112:7822-7830) and fluorescence resonance energy transfer (FRET) methods (Weiss, Science 1999, 283:1676-1683) have been developed on the single molecule level, many more experiments are now possible. Unfortunately in all single molecule studies, researchers are relegated to artificial fluorescent labeling of proteins of interest and limited to in vitro observation. Re-introduction of labeled proteins into the cell has yet to produce viable in vivo single molecule signals due to the large autofluorescent background and poor photostability of organic fluorophores.
Single molecules also have many inherently undesirable properties that limit the timescales of experiments. Excitation rates must be very high to yield biologically relevant information with reasonable time resolution (ms to seconds) and good signal to noise. Because the absorption cross-section (i.e. extinction coefficient, ε) of the best organic fluorophores is only ˜10−16 cm2 (i.e. ε˜105 M−1 cm−1) at room temperature (Macklin et al., Science 1996, 272:255-258), high intensity laser excitation must be utilized for single molecule fluorescence studies. Additionally, organic molecules can only withstand ˜107 excitation cycles before they photochemically decompose (Dickson et al., Nature 1997, 388:355-358; Lu & Xie, Nature 1997, 385:143-146; Macklin et al., Science 1996, 272:255-258). At 106 excitations/second (using ˜5 kW/cm2 excitation intensity and a typical collection/detection efficiency of 5%), this limits the time resolution to ˜1 ms (with an idealized signal to noise ratio of ˜7), and the average total time to follow an individual molecule before photobleaching of ˜10 seconds. While this can be a very large amount of data on very biologically relevant timescales, many of the excitation cycles end up being consumed by finding the molecules of interest before collecting data. Clearly, while reduction of oxygen can often increase the time before photobleaching, the photostability and overall brightness of the organic dyes limit all biological single molecule experiments. Thus, advances in fluorophore properties will be crucial to the continued success of all single molecule optical studies in biological systems.
Requiring similar coupling chemistry to that of organic fluorophores, water soluble II-VI quantum dots have recently been proposed and demonstrated as biological labels (Bruchez et al., Science 1998, 281:2013-2016; Chan & Nie, Science 1998, 281:2016-2018; Zhang et al., Analyst 2000, 125:1029-1031). Materials such as CdSe with protective and stabilizing ZnS overcoatings have size dependent optical properties and can be synthesized with very narrow size distributions (Murray et al., Z. Phys. D-Atoms Mol. Clusters 1993, 26:S231-S233; Murray et al., J. Am. Chem. Soc. 1993, 115:8706-8715; Peng et al., Nature 2000, 404:59-61). The strong absorption, spectral stability, and size-tunable narrow emission of these nanomaterials suggest exciting possibilities in biolabeling once further chemistry on the outer ZnS layer is performed to make these materials water-soluble (Rodriguez-Viejo et al., J. Appl. Phys. 2000, 87:8526-8534; Dabbousi et al., J. Phys. Chem. B 1997, 101:9463-9475). Because surface passivation is incredibly important in overall quantum dot optical properties, much care must be spent on quantum dot surface passivation and derivitization such that they can be reproducibly conjugated to proteins with predictable optical responses (Bruchez et al., Science 1998, 281:2013-2016; Chan & Nie, Science 1998, 281:2016-2018; Rodriguez-Viejo et al., J. Appl. Phys. 2000, 87:8526-8534; Dabbousi et al., J. Phys. Chem. B 1997, 101:9463-9475; Nirmal & Brus, Acc. Chem. Res. 1999, 32:407-414; Nirmal et al., Nature 1996, 383:802-804). In fact, successful implementation of water solubilization and surface passivation are only now beginning to bear fruit (Dubertret et al., Science 2002, 298:1759-1762; Jaiswal et al., Nat. Biotechnol. 2003, 21:47-51; Wu et al., Nat. Biotechnol. 2003, 21:41-46; Sutherland, Curr. Opin. Solid State Mat. Sci. 2002, 6:365-370; Gao et al., J. Biomed. Opt. 2002, 7:532-537; Mattoussi et al., J. Am. Chem. Soc. 2000, 122:12142-12150).
While quite promising due to their bright and very narrow size-dependent emission, multiple problems with using CdSe as biological labels still exist. Their synthesis requires high temperature methods using highly toxic precursors, they are comparable to the size of proteins that they may label (2-6 nm in diameter), and they suffer from the same need to externally label proteins of interest and possibly re-introduce the labeled proteins into cells. Thus, while the strong oscillator strengths enable quantum dots to be easily observed with weak mercury lamp excitation, thereby avoiding much of the more weakly absorbing autofluorescent background, they are still not an ideal solution to in vivo or in vitro single molecule studies.
Ideally, one would want the smallest possible genetically programmed label to be expressed on or adjacent to the protein of interest. Such an ideal label would need to have sufficiently strong absorption and emission as well as outstanding photostability to enable long time single molecule observation with high time resolution, even in the presence of high background fluorescence. Such a fluorescent probe does not yet exist. Currently the best available options due to being composed solely of amino acids, green fluorescent protein (GFP; Dickson et al., Nature 1997, 388:355-358; Heim, Proc. Nat. Acad. Sci, USA 1994, 91:12501-04; Ormo et al., Science 1996, 273:1392-5; Chattoraj et al., Proc. Nat. Acad. Sci, USA 1996, 93:362-67; Brejc et al., Proc. Nat. Acad. Sci, USA 1997, 94:2306-11; Cubitt et al., Trends in Biochem. Sci. 1995, 20:448-55; Kain & Kitts, Methods Mol. Biol. 1997, 63:305-24) and DsRed (Gross et al., Proc. Natl. Acad. Sci., USA 2000, 97:11990-11995; Jakobs et al., FEBS Lett. 2000, 479:131-135; Wall et al; Nat. Struct. Biol. 2000, 7:1133-1138; Yarbrough et al., Proc. Natl. Acad. Sci., USA 2001, 98:462-467) are excellent in vivo labels, and have been observed on the single molecule level in in vitro studies by many authors (Dickson et al., Nature 1997, 388:355-358; Malvezzi-Campeggi et al., Biophys. J. 2001, 81:1776-1785; Garcia-Parajo et al., Proc. Natl. Acad. Sci., USA 2000, 97:7237-7242; Cotlet et al., Chem. Phys. Lett. 2001, 336, 415-423; Lounis et al., J. Phys. Chem. B 2001, 105:5048-5054; Garcia-Parajo et al., Pure Appl. Chem. 2001, 73:431-434; Garcia-Parajo et al., ChemPhysChem 2001, 2:347-360; Garcia-Parajo et al., Proc. Natl. Acad. Sci., USA 2001, 98:14392-14397; Blum et al., Chem. Phys. Lett. 2002, 362:355-361).
In one of the first such studies, GFP's blinking and optical switching abilities have been studied as photons were used to shuttle the GFP chromophore between two different optically accessible states (Dickson et al., Nature 1997, 388:355-358). Unfortunately, while GFP can be specifically attached to the N or C terminus of any protein and expressed in vivo as a highly fluorescent label, problems, especially on the single molecule level, still remain. GFP is 27 kD or ˜4 nm in diameter (Ormo et al., Science 1996, 273:1392-5; Cubitt et al., Trends in Biochem. Sci. 1995, 20:448-55), and can therefore be a large perturbation to the protein to which it is attached. In addition, emission only occurs once GFP has folded into its final conformation, a process that can take up to ˜1 hour and, while examples of GFP labeling have been reported within all regions of different cells, sometimes GFP does not properly fold under a given set of conditions (Heim, Proc. Nat. Acad. Sci, USA 1994, 91:12501-04; Cubitt et al., Trends in Biochem. Sci. 1995, 20:448-55). Additionally, when considering single molecule studies, its emission significantly overlaps with the autofluorescent background, but its emission intensity is only comparable to standard exogenous organic dyes, thereby making in vivo single molecule studies very challenging. While largely insensitive to oxygen, it also typically bleaches after ˜107 excitation cycles, similar to standard organic dyes (Dickson et al., Nature 1997, 388:355-358). DsRed partially circumvents the issue of overlap with autofluorescent background, but while the red-shifted emission of DsRed relative to that of GFP could be an advantage, its comparable fluorescence intensity and tendency to form quadruplexes even at extremely low concentrations may further limit its use as an ideal biological label (Lounis et al., J. Phys. Chem. B 2001, 105:5048-5054; Garcia-Parajo et al., ChemPhysChem 2001, 2:347-360; Verkhusha et al., J. Biol. Chem. 2001, 276:29621-29624; Sacchetti et al., FEBS Lett. 2002, 525:13-19). Thus, the ideal label would combine the strong absorption, emission, and photostability of inorganic quantum dots, the small size and simple attachment chemistry of organic dyes, or preferably, like GFP and DsRed, the ability to be expressed in vivo as a single molecule biological label attached to any protein of interest without first purifying, labeling, and re-injecting the protein.
In summary, while current labeling methods and materials have enabled myriad bulk studies and many in vitro single molecule experiments, single molecule experiments remain limited by the disadvantages of even the best fluorescent probes. There is a need in the art for new single molecule probes, created with greatly improved photostability, much stronger absorption and emission under weak illumination, facile synthesis and conjugation to proteins, and tunable emission color. Ideally, such fluorescent labels should also be genetically programmable such that proteins under study can be directly labeled intracellularly, without first being over-expressed, purified, labeled, and then reintroduced into cells.